Transgenic/knockout approaches designed to disrupt signaling pathways via both loss-or-gain-of-function mutations have recently identified key pathways involved in regulating skin and appendage development. However, the exact roles that these genes/pathways play in normal skin- appendage development remain to be determined. The components of this Program Project focus on genes known to affect skin/appendage morphogenesis from the earliest known stage, to the very last stage of development. For example, p634 (Project 1) is expressed at E9.5 in the mouse, which is prior to epidermal stratification and appendage formation. In the absence of p63, the epidermis does not develop past an undifferentiated single-layered epithelium, which lacks appendages. Hair follicle formation is initiated at E14.5. Signaling via Tabby/Downless/Crinkled (Project 2) appears to determine whether an epidermal keratinocyte will become a hair follicle. Once this decision is made, both the sonic hedgehog (Project 3) and TGFbeta/BMP pathways and their downstream signaling mediators, the Smads (Project 4) are required for follicle maturation, however the exact sequence of events remains to be determined. The very final stage in development of the epidermis is the formation of carrier function. Based on several knockout models, it is now apparent that evolution has provided this pathway with several options to ensure that barrier formation occurs. Defects in barrier function resulting from the deletion of critical genes induces a compensatory response which activates the expression of replacement components (Project 5). Surprisingly, this occurs in utero. The molecular mechanisms that allow an embryo to compensate for a defect in barrier function in utero are unknown. The relevance of these projects to skin/appendage development is illustrated by the that all of the genes/signaling pathways to be studied have been shown to cause either inherited disorders or cancer when they are genetically altered in man. To overcome major limitations of conventional transgenic/knockout approaches, such as embryonic/neonatal lethality resulting from germline deletions (e.g., sonic hedgehog, patched, p63) or constitutive expression of transgenes (e.g., TGFbeta1, Smad7), recently developed inducible transgenic/knockout systems will be utilized (Cores B and C). The use of these models, coupled with in situ hybridization and cDNA array analysis (Core D), will identify the genetic events that occur in a spatio- temporally controlled manner during skin/appendage development. It is anticipated that this coordinated approach will provide a genetic blueprint of skin and appendage development.